GRK 1123:

Cellular Mechanisms of Learning and Memory Consolidation in the Hippocampal Formation

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This Research Training Group is funded by the German Research Council DFG

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©2006, FU Berlin
GRK 1123

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AG Ahnert-Hilger	AG Behr	AG Geiger	AG Haucke	AG Heinemann/ Kempter
AG Multhaup	AG Wulczyn	AG Rosenmund	AG Schmitz/ Brecht	AG Sigrist

Prof. Dr. Stephan Sigrist
Institut für Biologie, Genetik
Freie Universität BerlinBerlin
Takustr. 6
14195 Berlin
stephan.sigrist@fu-berlin.de

Topic

Molecular architectures of hippocampal mossy fiber bouton active zones

Title

Developmental and plasticity-related changes of active zones at hippocampal mossy fiber synapses

Issues of the project

Mossy fiber synapses are characterized by plasticity processes induced and executed at the presynaptic site. Thus, long-term changes of their presynaptic active zone architectures are likely to support their role long-term memory formation. We here seek to follow the assembly and disassembly of identified mossy fiber active zones with molecular and ultrastructural resolution. This way, we ask whether plasticity-related changes build on the “developmental” turnover of the active zones, which recently we observed at these synapses in cultured slice preparation.

Current State of Research

Several lines of evidence suggest that mossy fiber synapses play a key role in processing, long-term storage, and recall of spatial information in the hippocampal network (Lisman, 1999). Mossy fiber synapses show a rich repertoire of plastic properties, which are dominanted by contributions of the presynaptic site (Schmitz and Nicoll, 2005), Moreover, new formation of mossy fiber active zones can be related with spatial long-term memory formation (Ramirez-Amaya et al, 2001).

Active zones are the sites where the fusion of transmitter-filled vesicles takes place. Speed and accuracy of synaptic vesicle release are without precendence at other cellular locations. Active zones are characterized by macromolecular architectures often observable with electron microscopy (“dense bodies”, also known as cytoplasmic active zones ((Garner et al., 2002; Zhai and Bellen, 2004)). While number and “architecture” of active zones are likely of pivotal importance for presynaptic long-term plasticity, identification of AZ proteins has lagged behind the characterization of ion channels and synaptic vesicle proteins and fundamental components of active zones still await a functional characterization. Moreover, assembly and disassembly processes of active zone remain uncharacterized. Because AZs are typically about 200 nm in diameter, electron microscopy or high-resolution light microscopy are needed for their characterization.

• Garner et al (2002). Trends Neuroscience 25, 243-51
• Lisman et al (1999). Neuron 22, 233–242
• McBain (2008). Progress Brain Research 169, 225-40.
• Ramirez-Amaya et al. (2001). J. Neuroscience. 21, 7340-8.
• Schmitz and Nicoll (2005). Nature Review Neuroscience 6, 863-76
• Zhai and Bellen (2004). Physiology 19, 262-70

Previous work of the group

Synapses form by an asymmetric association of highly specialized membrane domains: at the presynaptic active zone transmitter filled vesicles fuse, while transmitter receptors at the opposite postsynaptic density sense this signal. Active zones are often associated with macromolecular electron dense assemblies (“dense bodies”) of largely unknown functionality.

We have been starting with analyzing neuromuscular synapses of Drosophila, similar to central mammalian synapses in molecular und ultrastructural composition. We recently identified a large coiled coil domain protein - Bruchpilot - a protein with homology to mammalian active zone component ELKS/CAST/ERC. Bruchpilot we showed to be a master organizer of synaptic active zones (Wagh et al., 2006). At Bruchpilot lacking active zones, electron-dense projections (T-bars) were entirely lost, Ca2+-channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. Bruchpilot-like proteins seem to establish proximity between Ca2+-channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel et al., 2006; also see „perspective“ in same issue of Science by Harold Atwood: Science 312, 1008-9).

Over the last years, we have been using cultivated organotypical slices of hippocampus to study the assembly of active zones at mossy fiber boutons with molecular resolution by expressing fluorescence-labeled CAST. The “molecular architectures” of active zones were found to show a remarkable degree of turnover, with active zone shape and size changing within few hours or less.

Our group capitalizes on use recent advances of light microscopy to study the mechanisms organizing assembly and plasticity of synapses under in vivo settings. We established protocols to study molecular dynamics during synapse assembly and plasticity in living animals, combining confocal und 2-photon in vivo (Rasse et al., 2005; Schmidt et al., 2008; Füger et al., 2008). Here, „Fluorescence recovery after photobleaching“ (FRAP) und photoactivation-experiments allowed us to quantify in vivo mobility of relevant synaptic proteins at identified populations of synapses.

Light microscopic inspection of synaptic organization is often restricted by the limited resolution of conventional light microcopy due to diffraction. To nonetheless further explore molecular substructures of synapses the group adapted a recent advance in high-resolution light microscopy: stimulated emission depletion microscopy (STED, s. Kittel et al., 2006 and references herein). STED breaks the diffraction barrier and allows localization of proteins well below 100 nm („in situ biochemistry“). Using STED, we observed BRP in doughnut-shaped structures centered at active zones of neuromuscular synapses, shining first light on the underlying macromolecular “architectures” pivotal for active zone structure and function.

• Füger, P. et al (2007). Nature Protocols 2, 3285-3298
• Kittel, R. et al. (2006). Science 312, 1051-1054
• Rasse, T et al (2005). Nature Neuroscience 8, 898-905
• Schmid, A. et al (2008). Nature Neuroscience 11, 659-666
• Wagh, D. et al. (2006). Neuron 49, 833-844

Objectives

Based on our results that active zones of mossy fiber synapses are subject to constant turn-over, we wish to analyze whether long-term plastic changes (evoked by tetanic stimulation, application of cAMP-analoga) quantitatively shift the distribution of active zone architectures between mossy fiber synapses.

In particular, the following questions should be addressed:

• After plasticity treatment, will there be more synapses with enlarged active zone architecture or will the amounts or the turnover of active zone components (as e.g. CAST, Rim, Ca2+- channels) change?
• Which precursor structures (vesicles) conribute to active zone assembly at mossy fiber synapses?
• How do changes in the molecular architecture to changes in release probability and short-term synaptic plasticity at mossy fiber active zones?
• How does presynaptically excuted long-term plasticity of mossy fibers relate to presynaptically excuted long-term plasticity of Drosophila synapses?

Methods

• Organotypic slice culture; confocal, 2-photon and high resolution light microscopy; electron microscopy; genetics of Drosophila and mouse; electrophysiological techniques; histology; transfections
• Data Analysis (Imaris; Amira), Modeling

Topics for Thesis Projects

• Quantitative description of active zone dynamics at hippocampal mossy fiber synapses using long-term imaging of cultivated slices
• Molecular sequence of active zone assembly at hippocampal mossy fiber synapses
• Structure-function relation of active zones at hippocampal mossy fiber synapses
• Comparing and complementing analysis of presynaptic long-term plasticity between Drosophila and mouse

Cooperation with other Members of the Graduate School  

• AG Schmitz/Brecht: With the group of Schmitz/Brecht we are characterizing active zone plasticity at mossy fiber boutons
• AG Geiger: With the Geiger group, Ca2+ current recordings from individual mossy fiber boutons will be related to active zone architectures
• AG Rosenmund: We will use autaptic cultures of granule cells for the analysis of active zone function changes (e.g. release probability)

Scholarship Holder:

• Elena Knoche (since June 2009)

 

AG Ahnert-Hilger	AG Behr	AG Geiger	AG Haucke	AG Heinemann/ Kempter
AG Multhaup	AG Nitsch/ Wulczyn	AG Rosenmund	AG Schmitz/ Brecht	AG Sigrist